Inhibition of IL-2-induced Akt phosphorylation and cell growth by LY294002 but not by AG490 and PD98059. KHYG-1 cells at an initial concentration of 2.5 × 10⁵/mL were cultured in the absence of IL-2 for 12 hours, rescued with IL-2 (100 U/mL) supplementation (IL-2^-→+), or treated with various concentrations of LY294002, AG490, or PD98059 for 1 hour in the absence of IL-2 before IL-2 supplementation. (A) At the indicated times after IL-2 supplementation (0, 5, 10, and 30 minutes), the protein levels of phosphorylated Akt kinase (Serine 473) and Akt kinase were determined by Western blot analysis. Equal loading of the samples was confirmed by protein levels of Akt. (B-C) Cells were treated with the indicated concentrations of LY294002 (B: 30 μM; C: 0, 10, 20, 30, and 50 μM) for 1 hour in the absence of IL-2 and harvested at the indicated times (B: 0, 5, 10, and 30 minutes; C: 5 minutes) after IL-2 supplementation. Western blot analysis was performed for Akt and phosphorylated-Akt. The ratio of phosphorylated-Akt to Akt was calculated by National Institutes of Health (NIH) image software and shown at the bottom of each panel. The results were representative of 3 independent experiments. (D-E) Cells were treated with the indicated concentrations of LY294002, AG490, and PD98059 for 1 hour in the absence of IL-2 and harvested 0, 24, and 36 hours after IL-2 supplementation. Viable cell numbers were assessed by a trypan blue dye exclusion method. The bars indicate 1 SD. The significance of cell number was determined by ANOVA test. ^*P < .01; ^**P < .05; #P > .05.